Extraction and Identification of Antibacterial Secondary Metabolites from Marine Streptomyces sp. VITBRK2

Actinomycetes were isolated from marine sediment samples collected from the east coast of Chennai, Tamil Nadu, India. Well diffusion and agar plug methods were used for the evaluation of antibiotic production by these isolates against drug resistant Methicillin- resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE). The potential isolate VITBRK2 was mass cultured for morphological and physiological characterization. The culturing conditions of the isolate were optimized and the recommendations of International Streptomyces Project were followed for the assimilation of carbon and nitrogen sources. The isolate was identified by comparing the properties with representative species in the key of Nonomura and Bergey’s Manual of Determinative Bacteriology. Ethyl acetate extract prepared from the cell free culture broth of the isolate was analyzed using HPLC- diode array technique to characterize the metabolites and identify the antibiotics. VITBRK2 was found to be Gram-positive rod grey color aerial mycelium production. It was also non motile in nature with spiral spore chain morphology. VITBRK2 was identified as Streptomyces and designated as Streptomyces sp. VITBRK2. HPLC-DAD analysis showed the presence of indolo compounds (3- methyl-indole and 2-methyl- indole) along with amicoumacin antibiotic. The observed activity of Streptomyces sp. VITBRK2 against MRSA and VRE strains may be due to the presence of indolo compounds in the isolate. The results of this study suggested that secondary metabolites produced by Streptomyces sp. VITBRK2 could be used as a lead to control drug resistant bacterial pathogens.

developed resistance to the majority of conventional antibiotics (3). For more than two decades, these strains were controlled with vancomycin. However, there is an increased incidence of emergence of antibiotic-resistant strains (4,5). The problem of antibiotic resistance is further complicated by the emergence of other  (11)(12). Currently, many scientists are working on new antimicrobial drugs, mainly of actinomicetal origin (13). Actinomycetes are producers of metabolites with antimicrobial, anti-parasite, antiviral, antitumor activity, cytotoxic, etc; whose chemical structures are unique (14).
Until recently marine sediments as a source of bioactive actinomycetes have remained as the least explored resources, but today it becomes one of the more promising sources. Streptomyces are a prolific source of secondary metabolites yielded many antibiotics; more than 80% of antibiotics available in the market are from Streptomyces (15). In the present study, we report the antibacterial activity of Streptomyces isolated from marine sediments against MRSA and VRE strains.

Screening for antibiotic production
Antibacterial activity of the potential isolate was studied by agar plate diffusion assay. Briefly, filter disks (6mm in diameter) (16). Inhibition zones were expressed as diameters and measured after incubation at 37°C for 24h. All actinomyces isolates were screened for antibacterial activity against all seven ATCC drug resistant strains.
Influence of the various culture media on the antibacterial potential of the isolate was studied by cylinder plug method using ISP 1 supplemented with seawater collected at the sampling site, marine agar, actinomycetes isolation agar, starch casein agar (Himedia, Mumbai, India).

Characterization and identification of the potential isolate
The morphological, cultural, physiological and biochemical characterization of the potential isolate was carried out as described in ISP (17). The morphology of the spore bearing hyphae with the entire spore chain with the substrate and aerial mycelium of the strain was examined by light microscope (1000x magnification) as well as scanning electron microscope (Hitachi, S-3400N).
Media used were those recommended in the ISP (18). Mycelium was observed after incubation at 28°C for 2 weeks and colors were also determined.
Carbohydrate utilization was determined by growth on carbon utilization medium (ISP 9) (19) supplemented with 1% carbon sources at 28°C.
Temperature range for growth was determined on inorganic salts starch agar medium (ISP 4) using a temperature gradient incubator. Hydrolysis of starch and milk were evaluated by using the glucose starch agar and skim milk agar, respectively.
Reduction of nitrate and production of melanin pigment were determined by the ISP method (20).
All cultural characteristics were recorded after 14 days.

Optimization of nutritional and cultural conditions
In order to optimize the nutritional and

HPLC-DAD analysis of the EA extract
A total volume of 15 litters of the culture broth was centrifuged in batches for 15 min at It was observed that the mature sporulating aerial mycelium was whitish grey in color. The spore chain morphology was observed under optical microscope at 1000X magnification. The smooth spore surface morphology was observed under scanning electron microscopic (SEM) analysis ( Fig.1). The growth of the isolate was maximal on ISP1 medium supplemented with sea water and its growth was equally maximal on actinomycetes isolation agar as well. The isolate showed maximum growth when cultivated at temperature 28°C; pH 7.4, with seawater 25%. The isolate assimilated arabinose, xylose, inositol, mannitol, fructose, sucrose and raffinose, however, the isolate did not utilize rhamnose (Table.2).